The glutathione-S-transferases (GST) are found in nearly all forms of life. These ubiquitous multifunctional proteins catalyze the nucleophilic addition of glutathione to a wide variety of lipophilic reactive species including insecticides and herbicides. GST has also been implicated in the development of resistance to pesticides. Although considerable information is available on the substrates for GST catalyzed glutathione conjugation, current knowledge enzyme structure and mechanism of action remains limited. The overall objectives of this proposal are to fully define the stereochemical factors involved for effective binding of an electrophilic substrate within the enzyme H-site as well as the binding of glutathione analogs within the G-site. These data will be incorporated into the design of isozyme class specific inhibitors of GST. Ultimately, the goal of this project is to develop an irreversible in- vivo isozyme class specific inhibitor. The stereochemical features of H- and G- sites will be probed for Alpha and Mu class isozymes using unsaturated trifluoromethyl ketones, glutathione conjugates of fluoroketones, peptide analogs of glutathione bearing pendent electrophilic functionality, and vinyl fluoride peptide isosteres. Each series of inhibitors will be assayed against rat liver isozymes 1-1,2-2,3-3 and 4-4. The stereoselectivity of the conjugation reaction (enzyme catalyzed) will be assessed by spectral and chemical degradation studies. The development of isozyme class specific inhibitors would significantly enhance the basic understanding of GST catalysis, specifically in the area of pesticide toxicity. Specific inhibitors will provide a means to assess the relative contribution of individual GST isozymes to in-vivo pesticide metabolism and detoxication as well as their involvement in the development of resistance. In addition to the metabolic and detoxifying functions, GST isozymes play a major role in intracellular transport. Inhibitors of GST may be useful in establishing the contribution of specific isozymes to pesticide transport.